Electrophoretic method for preparative separation of charged molecules in liquids

ABSTRACT

The method for electrophoretic transport of at least one solute from a first solvent stream to a second solvent stream utilizes an apparatus having a housing, a pair of electrodes positioned with the housing, each electrode being positioned adjacent one of a pair of opposite ends of the housing and each being surrounded by a device to provide a flow of a buffer around the electrode, a first inlet device through which the first solvent stream is introduced into the housing and an associated outlet device, a second inlet device through which the second solvent stream is fed into the housing and an associated outlet device, a plurality of electrophoretic membranes extending in substantially parallel, space apart, array transversely of the housing and between the electrodes, the membranes being structured to define a first solvent path for the first solvent stream and a separate second solvent path for the second solvent stream. To help prevent membrane clogging the method includes the steps of stopping or reversing the voltage across the electrodes during the electrophoretic separation while maintaining a new movement of the solute species in the desired direction from the first solvent stream to the second solvent stream and while maintaining the first solvent stream in the first solvent path and the second solvent stream in the second solvent path.

TECHNICAL FIELD

The present invention relates to methods of, and apparatus for,preparative electrophoresis and in particular to such methods andapparatus which may be used in the large scale recovery of solutemacromolecule.

BACKGROUND ART

Preparative electrophoretic separation of proteins and other compounds,i.e. separation by charge within an electric field, is normally carriedout in a column of anticonvectant medium, e.g. chemical gel. The samplesare delivered to the upper surface of the gel, electric potential isapplied and, after a time interval, separated components emerge at thebottom of the gel column. The scale of this method of preparativeseparation is limited by the cross section of the gel which must notexceed the capacity of the system to remove heat generated by thecurrent. This cross section can only be of the order of several squaremillimeters because of the very poor heat conductivity of the gel. Thepresent invention aims to overcome these limitations in a number ofways.

It is also known to carry out electrophoretic separation as between freeflowing streams either with free or fixed boundaries. In free boundaryelectrophoretic separation macromolecular species in solution, includingcolloidal solution, are separated as a stream of the solution is passedbetween charged electrodes. The stream is then divided, withoutremixing, into a plurality of parts with differing proportions of themolecular species present in the original stream (see U.S. Pat. No.2,878,178, and British Patent specification 1,255,418). In fixedboundary electrophoretic separation a semi-permeable membrane acts as afilter through which the liquid stream passes (see U.S. Pat. No.3,079,318) or acts to separate two streams of liquid between which atleast one molecular species migrates under the electrophoretic influence(see PCT Patent specification W079/00002 and U.S. Pat. No. 3,989,613).

A problem with fixed boundary electrophoresis is the fouling or cloggingof the membranes. The efficiency of the process depends upon:

1) maximum contact of the processed solution with the membrane surface,and

2) unimpaired permeability of the membrane pores intended to permit thepassage of the macromolecules in question.

Maximum contact, in turn, depends on uniform distribution of fluid flowin the spaces between the membranes. Because of the relatively slowpassage of the fluid in electrophoretic separation manifolding of theflow through parallel spaces in a stack of electrophoretic membranes aswell as ensuring a uniform flow of the fluid film along each space maybe difficult because of preferential channelling due to slightdifferences in the geometry, air locks and similar imperfections. Thepresent invention aims, in a first aspect, to overcome this problem.

It will be realised that membrane fouling or clogging can occur in fixedboundary electrophoretic separation when the pores in the membranesapproach in size the molecules being separated. In a second aspect thepresent invention aims to overcome this problem.

A further problem with known fixed boundary electrophoretic separationhas been the difficulty in maintaining a free flow of solvent betweenadjacent membranes. In a third aspect the present invention providesmeans to effectively space adjacent membranes while allowing free flowof solute therebetween.

A still further problem with known fixed boundary electrophoreticseparation apparatus has been the physical problems of filling,emptying, cleaning and reassembling of a housing containing theelectrodes and membranes. Conventionally such a housing has been formedsubstantially integrally with associated storage tanks, pumps, coolingapparatus and the like making the housing itself difficult to handle. Ina fourth aspect the present invention provides improved apparatus forovercoming these physical handling problems.

SUMMARY OF THE INVENTION

The present invention consists in a method for causing theelectrophoretic migration of at least one solute from a first solventstream to a second solvent stream in an appartus comprising a housing, apair of electrodes positioned within the housing, each electrode beingpositioned adjacent one of a pair of opposed ends of the housing andeach being surrounded by means to direct a flow of a suitable bufferpast the electrode, first inlet means through which the first solventstream is introduced into the housing and outlet means therefor, secondinlet means through which the second solvent stream is introduced intothe housing and outlet means therefor, a plurality of electrophoreticmembranes extending in substantially parallel, spaced apart, arraytransversely of the housing and between the electrodes, the membranesserving to define a first solvent path for the first solvent stream anda separate second solvent path for the second solvent stream, the methodbeing characterized in that the voltage across the electrodes isperiodically stopped or reversed, while maintaining a net movement ofthe solute species in the desired direction from the first solventstream to the second solvent stream and while maintaining the firstsolvent stream in the first solvent path and the second solvent streamin the second solvent path.

In a preferred embodiment of the invention the solvent in the firstsolvent stream is caused to flow in a pulsitile fashion.

The pulsing of the first solvent stream may be brought about byintermittently occluding elastic tubing leading from a peristaltic pumpto or from the electrophoretic cell. The intermittent occlusion of suchelastic tubing may be brought about directly or indirectly by a suitablecam, eccentric or like means. In an alternative preferred embodiment apump, preferably on the downstream side of the electrophoretic cell isintermittently actuated or operated at differential speeds to producethe required pulsatile flow in the first solvent stream.

The operation of the abovementioned preferred embodiment of theinvention is most easily understood with reference to a cell having onlythe first and second solvent streams, the first solvent stream being, sofar as the solute molecules are concerned, upstream of a separatingmembrane and the second solvent stream being downstream thereof. In suchan arrangement five phases can be discerned in the electrophoreticmigration:

1) Upstream fluid phase;

2) Upstream interface with membrane;

3) Membrane gel;

4) Downstream interface;

5) Downstream fluid phase;

Optimal requirements for each of these phases are different:

1. Electrophoretic migration in the upstream fluid will be disturbed bya rapid turbulent flow. On the other hand a flow rate may be so slowthat all the relevant components will migrate to the proximal part ofthe membrane, and a large distal part of the membrane may notparticipate in the process. Thus a preferred flow pattern in this phaseis stepwise with one step following: designed to replace quickly thespent solution with a fresh one, followed by a stationary period toallow migration to the membrane. The optimal volume and duration of eachcycle will depend on the prevailing conditions.

2. The upstream fluid/membrane boundary may be blocked by obstructedmolecules forming an insoluble film on the surface, a condition called"fouling" in filtration processes. When necessary this can becounteracted by temporarily reversing the electric field toward the endof each cycle to detach the aggregating molecules. Frequency andduration of field reversal must be determined experimentally and may becontrolled manually or automatically by monitoring changes in electricalconductivity of the stack during each phase or cycle.

3. Within the membrane the transport is dependent mainly on thecharacteristics of current and membrane composition and is isolated fromthe flow mechanics.

4. In the downstream fluid phase a vigorous cross flow is desirablebecause it prevents molecules adhering to limiting membranes separatingthe solvent from the buffer streams and offers the opportunity for heatremoval with the aid of an external heat exchanger.

The pulsatile flow will, to some extend, reduce surface fouling of themembranes, however, additionally the shape of the electric fieldaccording to this invention can prevent membrane occlusion. While themechanism whereby the periodic reversal of the polarity of theelectrophoretic cell is quite complex and, among other factors, isinfluenced by brownian movement and adsorbtion onto the membrane, it isconsidered that the action can be likened to a sieve in which thescreening of particles is promoted by vibration of the sieve.

Adjacent membrane in the apparatus in which the present method iscarried out are preferably separated by a pair of substantially parallelscreens. In a preferred embodiment of the invention the parallel screensare woven and are so selected that the meshes thereof cannot nest withone another. Ordinarily the spaces between adjacent membranes aremaintained by intervening gaskets and mesh screens which prevent themembranes from adhering to each other. However, it was observed thatwithout the benefit of high positive pressure the membranes tend tocling to the surface of the mesh causing almost complete obstruction ofthe flow. The use of at least two adjacent screens to define the spacesbetween the membranes was found to greatly improve the flow. Furtherimprovement was introduced following the observation that parallelscreens of the same mesh size tend to partially nest into each othercausing an interference with a free flow. This is preferably preventedby ensuring that the mesh of adjacent screens is not identical. Forexample, a square mesh may alternate with a diagonal mesh or theadjacent screens may be made of different mesh size (e.g. with 0.3 and0.4-0.5 mm openings). Preferably the two screens are incorporated into asingle molded gasket to minimize handling.

In a conventional filter stack the fluid is forcibly pumped to create across flow in the spaces between membranes, the whole stack beingsandwiched between rigid walls to prevent disruption by the highinternal pressure. This configuration is not suitable forelectrophoresis because the support walls would interfere with theelectric field across the stack and with the removal of gases generatedby the electrodes. In the present invention the stack is preferably keptcollapsed without the aid of external supports by placing the pumps onthe downstream side i.e. on the downstream side of the electrophoreticcell, so that the flow is effected by suction, while the fluid entersthe stack passively to replace the fluid removed by the pumps. On theother hand the external buffer circulation is preferably maintained by apositive pressure which also helps to maintain the stack in a compressedstate. Alternatively, the compartments carrying the circulating buffermay be pressurized and the flow through the stack effected by a positivepressure feed which is kept lower than the external buffer pressure. Themaintenance of free flow and even distribution of fluid within the stackis essential for effective separation. Elongate, flexible inlet andoutlet tubes, preferably from the buffer and solvent streams, areprovided to connect the electrophoretic cell and supply and/or dischargetanks for the buffer and solvent streams. The inlet and outlet tubes arepreferably bound together to form a single umbilicus. This arrangementfacilitates the free movement of the cell relative to the remainder ofthe apparatus. The cell so connected to the remainder of the apparatusis preferably mounted so that it may be tilted in at least one planewhich facilitates filling and emptying the electrophoretic cell.

The method according to this invention is preferably carried out in anapparatus for the electrophoretic separation of molecular speciespresent in a liquid into at least two component groups, comprising ahousing, a pair of electrodes positioned within the housing, eachelectrode being positioned adjacent one of a pair of opposed ends of thehousing and each being surrounded by means to direct a flow of asuitable buffer past the electrode, a plurality of semi-permeablemembranes divided into at least two groups, the membranes of each groupextending in substantially parallel, spaced apart, array across thehousing intermediate the electrodes to form a plurality of chambers,each group of chambers being separated from adjacent groups of chambersby barriers which do not interrupt the passage of an electric currentbetween the electrodes but will effectively prevent liquid flowtherethrough, an inlet into each of the chambers of one group thereof,an outlet from each of the chambers of one group thereof, each chamberof one group being connected in parallel or in a series with acorresponding chamber of the, or each, other group such that liquid maypass therethrough from the respective inlet to the respective outlet.

In one embodiment of this apparatus the membranes in each group diminishin pore size in the direction of migration of the molecular speciesbeing spearated. Normally all of the molecular species to be separatedwill be introduced into a first liquid stream flowing through a firstchamber of each group. The molecular species will pass, under theinfluence of the electrophoretic potential into a second, third, etc.,one of the chambers according to the respective molecular size of eachof the species. It is also possible however to introduce the molecularspecies into the liquid stream flowing through two or more of thechambers of each group.

The chambers are preferably formed by combined gaskets and screenspositioned between adjacent membranes. The inlet and outlet passages andthe passages interconnecting the corresponding chambers of the variousgroups passing through the abutted membranes and gaskets.

The membranes used in the methods and apparatus according to the variousaspects of the present invention are preferably formed by apolyacrylamide gel. One advantage of polyacrylamide gel as a matrix forelectrophoretic separation is the ease of manufacture and of controllingthe pore size within wide limits. However, the polymer is too friable tobe handled as thin membranes. This can be overcome by making use ofsheets of porous material, such as paper or fabric impregnated withpolyacrylamide.

In the present invention the electrodes are preferably surrounded by ajacket of flowing buffer which is pumped to and from a distant tank ortanks, heat and gas bubbles being removed in the process. The electrodesmay be made of a thin platinum wire but plate or foil of a suitablematerial could be used. The buffer may be separated from the liquidstream containing the molecular species to be separated by a membrane ofsufficiently small pore size. Alternatively, in the case of wireelectrodes, the electrodes may be encased individually by hollow fibresor tubes of semipermeable material carrying the circulating buffer.

Unless a complete separation can be accomplished in a single passage,the feed solution can be recirculated. In this case it is desirable toprevent mixing of the weaker return solution with the richer residue inthe reservoir. A "raft" floating on the surface of the residual fluidcan be used to dissipate the kinetic energy of the returning streamwhich will then be layered gently above the residual solution. Inaddition some kind of anticonvectant packing (e.g. beads, sponge orchain) can be used in the recirculation tank. Similarly, recirculationof the downstream effluent can be used for progressive concentration ofthe extracted components.

Preferably the upstream flow is intermittent and involves a nearcomplete exchange of the solution in the stack. This can be effected bya piston pump (e.g. a syringe) with a one way inlet and outlet valves.The pump is preferably positioned on the downstream side of the stack.Preferably the pump mechanism is linked with the current reversal switchto ensure synchronous operation as in the following example:

1. Stroke 1 (e.g. 5 seconds). Plunger out, pump filled with the solutionwithdrawn from the stack (replaced by fresh fluid sucked from thereservoir).

2. Interval (e.g. 60 seconds) for electrophoretic transport, while theplunger is being slowly depressed, expelling pump effluent to acollection or recirculation reservoir. Current may be reversed by alinked mechanism during the last few seconds of the cycle.

At the same time the downstream flow is controlled independently asdescribed above. It is important however to maintain the mean pressurewithin the stack less than that in the buffer solutions to maintain thestack in a compressed condition. Advantage may be had where one of thesolute macromolecular containing streams is caused to flow in apulsitile manner to momentarily cause the pressure therein to equal orjust exceed the pressure in the buffer solution.

The solutions of the macromolecular solute species may be true solutionsor colloidal solutions.

The various aspects of the present invention taken together orseparately assist in the separation of solute macromolecules on a muchlarger scale than has previously been possible. Proteins may, forinstance, be recovered in amounts exceeding 100 g/hr.

BRIEF DESCRIPTION OF THE DRAWINGS

Hereinafter given by way of example only is a preferred embodiment ofthe present invention described with reference to the accompanyingdrawings in which:

FIG. 1 is a diagrammatic perspective view of an electrophoreticapparatus according to the present invention;

FIG. 2 is a cross sectional view through II--II of FIG. 1;

FIG. 3 is a diagrammatic exploded view of a stack of the membranes foruse in the electrophoretic apparatus of FIG. 1;

FIG. 4 is a graph showing a number of possible variations in flow rateof the upstream and downstream fluids through the electrophoreticapparatus of FIG. 1; and

FIG. 5 is a diagrammatic longitudinal sectional view through a pump foruse in the electrophoretic apparatus according to the present invention.

FIGS. 6 and 7 respectively show diagrammatically a more complex array ofmembranes and gaskets for use in the electrophoretic apparatus of FIG.1.

BEST MODE OF CARRYING OUT THE INVENTION

The electrophoretic apparatus 10 includes a housing which containselectrodes and an array of electrophoretic membranes as will behereinafter described, sources of supply of an upstream liquidcontaining a solute, a downstream liquid and a buffer solution, suitablepumps and ducting and a source of electrical potential for causing thesolute to move electrophoretically across the intervening membranesbetween the upstream liquid and the downstream liquid.

The upstream liquid 11 is contained within an open topped container 12.The upstream liquid flows from container 12 through tube 13 to thehousing 14. It returns from the housing 14 through tube 15, pump 16 andtube 17 to the container 12. A floating pad of material 18 is providedin container 12 such that the returning, generally less dense, upstreamliquid introduced into container 12 does not mix unduly with the,generally more dense, upstream liquid already in the container 12.

The downstream liquid 19 is contained within an open-topped container21. The downstream liquid flows from container 21 through tube 22 to thehousing 14. It returns from the housing 14 through tube 23, pump 24 andtube 25. Cooling means 26 comprising a coil through which a refrigerantpasses is immersed in the downstream liquid 19 in container 21 to coolthe downstream liquid. Alternatively, or in addition, either or both ofthe upstream and downstream liquids could be passed through a coilimmersed in a cooling bath.

The buffer solution 27 is contained in an open topped container 28. Itis pumped by pump 29 through tube 31 to housing 14 from whence itreturns through tube 32 to container 28. Cooling means 26 includes acooling coil in container 28 to cool the buffer solution 27. Each of thetubes 31 and 32 is bifurcated adjacent the housing 14 such that aseparate buffer stream flows past each of the electrodes in the housing14.

A D.C. power supply 33 provides a source of electric potential to drivethe electrophoretic migration of solute molecules from the upstreamliquid into the downstream liquid. The power supply 33 is connected bywires 34 to terminals 35 on the housing 14.

The upstream liquid tubes 13 and 15, the downstream liquid tubes 22 and23, the buffer tubes 31 and 32 and the wires 34 are all bound togetherby bundle ties 36 to form a single umbilical cord 37. This allows readymovement of the housing 14 relative to the other components in theelectrophorectic apparatus 10.

The housing 14 is pivotably mounted on a stand 38 by a bolt 39 whichpasses through a hole in stand 38 and a corresponding hole in a tab 41extending from the underside of housing 14. Loosening of bolt 39 allowsthe housing to be pivoted about the axis of the bolt. This allows forlowering the inlet end of the housing 14 when the housing is first beingcharged with the upstream, downstream and buffer liquids. Such aprocedure helps reduce the development of air locks in the housing 14.Similarly the housing 14 may be more conveniently drained by depressingthe outlet end of the housing 14.

The housing 14 includes a rectangular top plate 42 having on itsundersurface a rectangular rib 43. Adjacent one end the top plate 42 isformed with vertical holes 44 and 45 which extend through top plate 42and rib 43 adapted to receive tubes 13 and 22 respectively while theother end of the tap plate 42 is similarly formed with holes (not shown)to receive tubes 15 and 23. A bore 46 extends horizontally into the topplate 42 adjacent the one end thereof and is adapted for connection toone arm of the tube 31. A side hole 47 connects bore 46 with theunderside of the top plate 42. Similarly a bore (not shown) is providedat the other end of the top plate for connection with one arm of thetube 32.

The underside of the top plate 42, within the area bounded by the rib 43is provided with a plurality of cleats 48 to hold in place a platinumelectrode wire 49 which is connected to one of the terminals 35.

The housing 14 further includes a bottom plate 51 which is similarlyprovided at one end with a bore 46 and a side hole 47 to introducebuffer solution 27 through one arm of tube 31 and at the other end witha further bore (not shown) for connection to buffer discharge tube 32;and with cleats 48 for electrode wire 49. The bottom plate 51 isprovided on its upper surface with a rectangular rib 52 having internaldimensions just larger than the outside dimensions of rib 43.

A rectangular spacer frame 53 completes the housing 14 and is disposedbetween the top plate 42 and bottom plate 51. The spacer frame closelysurrounds each of the ribs 43 and 52.

An array of boundary membranes 54, electrophoretic membranes 55 andspacers 56 are disposed in parallel planar array within the housing 14.The boundary membranes 54 are disposed at the top and bottom of themembrane stack and at is midpoint. The boundary membranes 54 are soconstructed that they prevent the passage of the solute macromoleculeswhile permitting the passage of an electric current carried by smallions of the buffer. At each end the bondary membranes are formed withholes in alignment with the holes 44 and 45 in the one end of top plate42 and the corresponding holes at its other end.

Each of the electrophoretic membranes 55 is so constructed to allowpassage of at least the desired solute molecule therethrough. Themembranes 55 are also formed at each end with holes in alignment withholes 44 and 45 in the one end of top plate 42 and the correspondingholes at its other end.

Each of the adjacent boundary membranes 54 and electrophoretic membranes55 is separated by a spacer 56 comprising a peripheral gasket 57surrounding, and formed integrally with, a pair of plastic mesh screens58 lying in parallel face to face array, the two screens 58 are eitherof different mesh size or the respective warps and wefts of the meshesare disposed at an angle to one another so that the two meshes cannotnest together. Each spacer is provided with two holes at each end tocorrespond to the holes in the ends of the membranes 54 and 55. One ofeach of these holes is however surrounded by the gasket. The correctarrangement of the screens keeps the upstream and downstream liquidstreams separate while allowing the two streams to flow along oppositeside of each of the electrophoretic membranes as shown in FIG. 3. Thearrangement shown in FIG. 3 provides for a pair of electrophoreticmembranes 55 operating in parallel. In alternative arrangements thegaskets may be constructed to provide for two or more groups ofmembranes, each group being subdivided by one or more electrophoreticmembranes to form a plurality of chambers which are joined in series orin parallel. If more than one electrophoretic membrane is provided ineach chamber, as is required if more than two solvent streams are beingused, the electrophoretic membranes may be of different pore sizes.Preferably the pore sizes diminish in the direction of migration of the,or one of the, solute species.

In operation buffer solution 27 is passed rapidly, at low pressure,through the spaces surrounding the electrodes 49 adjacent the top plate42 and bottom plate 51. This flow of buffer solution 27 serves to removeheat and gas bubbles from housing 14. The flow of downstream liquid 19is preferably continuous through the housing 14 as shown by flow line(a) of FIG. 4.

The flow of the upstream liquid 11 through the housing 14 is preferablycarried out in a pulsitile fashion. In embodiment (b) of FIG. 4 the flowis intermittently stopped by stopping pump 16 or by otherwise occludingthe flow. The sawtooth flow pattern of embodiments (c) and (d) may beachieved by a series of sudden compressions of the tube 13 or 15 e.g. bythe impact of a falling weight operated by a cam. The pulses will be inthe positive or negative direction depending on whether the pump is onthe upstream side (c) or downstream (d) of the housing 14. In each casethe net total flow is not altered. The optimum frequency, shape andamplitude of the pulses may be determined experimentally by routinetesting.

FIG. 5 shows a preferred pump 16 for pumping the upstream solvent 11through the housing 14. The pump 16 comprises a piston 62 moveable in acylinder 63 and biased outwardly therefrom by a spring 64. The cylinder63 is in fluid communication with a pair of one way valves shown at 65.The piston 62 is operated by rotation of a cam 66 as indicated. The cam66 slowly depresses the piston 62. As the piston approaches the end ofits travel it contacts switch 67 to reverse the polarity of theelectrodes. As the cam step passes beyond the piston 62 the springcauses the piston to draw backwards quickly. This rapid back movement ofthe piston 62 recontacts the switch to restore the normal polarity tothe electrodes and simultaneously causes a rapid flow of solvent throughthe housing 14.

A further development of flow distribution in the separation stack isillustrated in FIGS. 6 and 7, in which FIG. 6 shows three explodedsections through different planes (A,B,C). The 12 numbered drawings ofFIG. 7 show the sequence of the individual elements with the letters andnumbers corresponding to the ones in the sections. In this case thespaces are connected not by external tubing but by an internalarrangement of perforations in the membranes and gates in the gaskets.By way of example the stack has three inlets (in) and outlets (out)corresponding to three separate streams shown in the sections by arrowedlines. Also the sequence of membranes (e.g. of decreasing permeability),m₁, m₂ and m₃, and corresponding gaskets (g) is repeated once toincrease the surface area. The gaskets have three pairs of openings butonly one of these pairs is connected to the central space by gates(parallel lines in the plane view). The membranes (m₁,2,3) have matchingperforations, shown as circles in three positions selected so as to keepthe three streams separate by directing the flow through the appropriatespaces, e.g. A-through 2 and 8, B-through 4 and 10 and C-through 6 and12. It is to be understood that the number of streams can be increasedhorizontally e.g. by repeating the same sequence (in a larger version ofthe apparatus) to increase throughput or by adding new membranes withdiffering characteristics. The number of cycles of membranes etc. in thestack can also be multiplied as required to increase the scale ofseparation. It is also envisaged that pre-assembled modules can bemanufactured to be used in or included into a separation stack.

I claim:
 1. In a method for causing electrophoretic migration of atleast one solute from a first solvent stream to a second solvent streamin an apparatus comprising a housing, a pair of electrodes positioned inthe housing, each of the electrodes being positioned adjacent one of apair of opposite ends of the housing and each of the electrodes beingsurrounded by means to direct a flow of a buffer past the electrode,first inlet means through which the first solvent stream is fed into thehousing and associated first outlet means, second inlet means throughwhich the second solvent stream is fed into the housing and associatedsecond outlet means, a plurality of electrophoretic membranes extendingin substantially parallel, spaced apart, array transversely of thehousing and between the electrodes, the membranes being structured toform a first solvent path for the first solvent stream and a separatesecond solvent path for the second solvent stream, the improvementcomprising the step of periodically one of stopping and reversing avoltage applied across the electrodes, while maintaining a net movementof the solute from the first solvent stream to the second solvent streamand while maintaining the first solvent stream in the first solvent pathand the second solvent stream in the second solvent path.
 2. Theimprovement as defined in claim 1, further comprising the step ofproducing a pulsatile flow of the first solvent stream through thehousing.
 3. The improvement as defined in claim 2, wherein in the stepof producing the pulsatile flow of the first solvent stream through thehousing the first solvent stream is caused to proceed at a comparativelyfaster flow rate for a comparatively shorter time period and then at acomparatively slower flow rate for a comparatively longer time periodand the voltage applied across the electrodes is one of stopped andreversed near the finish of each of the comparatively longer timeperiods of the slower flow.
 4. The improvement as defined in claim 1,further comprising the step of separating each of adjacent ones of themembranes by a pair of substantially parallel screens, each of thescreens having a mesh.
 5. The improvement as defined in claim 4, whereinthe parallel screens are woven and so selected that the meshes cannotnest with each other.
 6. The improvement as defined in claim 1, furthercomprising the step of pumping the solvent streams through the housingat a pressure which is less than that of the buffer.
 7. The improvementas defined in claim 1, in which the apparatus used to perform the methodincludes a supply and discharge container for each of the first andsecond solvent streams and the buffer and further comprising the stepsof connecting the housing through a plurality of elongated flexibletubes to the supply and discharge containers for the first and secondsolvent stream and the buffer and binding the flexible tubes togetherinto a single umbilicus.
 8. The improvement as defined in claim 2,further comprising the step of dividing the membranes in the housinginto at least two groups, the membranes in each of the groups extendingin substantially parallel, spaced apart, array across the housingbetween the electrodes to form a plurality of chambers, each group ofchambers being separated from adjacent groups of chambers by barriers,said barriers not interrupting an electric current between theelectrodes but effectively preventing liquid flow therethrough, each ofthe chambers of one group being connected in parallel or in series witha corresponding chamber of the, or each other group of chambers suchthat the first and second solvent streams pass through arrays of thechambers.
 9. The improvement as defined in claim 1, further comprisingproviding more than two of the solvent streams and separating each ofthe solvent streams from an adjacent one of the solvent streams by oneof the membrane, the membrane separating the solvent streams having poresizes decreasing in a direction in which electrophoretic migration ofthe solute occurs.